Gregory M. Holmes

Vagally mediated effects of glucagon-like peptide 1: in vitro and in vivo gastric actions

Glucagon‐like peptide‐1 (GLP‐1) is a neuropeptide released following meal ingestion that, among other effects, decreases gastric tone and motility. The central targets and mechanism of action of GLP‐1 on gastric neurocircuits have not, however, been fully investigated. A high density of GLP‐1 containing neurones and receptors are present in brainstem vagal circuits, suggesting that the gastroinhibition may be vagally mediated. We aimed to investigate: (1) the response of identified gastric‐projecting neurones of the dorsal motor nucleus of the vagus (DMV) to GLP‐1 and its analogues; (2) the effects of brainstem application of GLP‐1 on gastric tone; and (3) the vagal pathway utilized by GLP‐1 to induce gastroinhibition. We conducted our experiments using whole‐cell recordings from identified gastric‐projecting DMV neurones and microinjection in the dorsal vagal complex (DVC) of anaesthetized rats while monitoring gastric tone. Perfusion with GLP‐1 induced a concentration‐dependent excitation of a subpopulation of gastric‐projecting DMV neurones. The GLP‐1 effects were mimicked by exendin‐4 and antagonized by exendin‐9–39. In an anaesthetized rat preparation, application of exendin‐4 to the DVC decreased gastric tone in a concentration‐dependent manner. The gastroinhibitory effects of exendin‐4 were unaffected by systemic pretreatment with the pro‐motility muscarinic agonist bethanechol, but were abolished by systemic administration of the nitric oxide synthase (NOS) inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME), or by bilateral vagotomy. Our data indicate that GLP‐1 activates selective receptors to excite DMV neurones mainly and that the gastroinhibition observed following application of GLP‐1 in the DVC is due to the activation of an inhibitory non‐adrenergic, non‐cholinergic input to the stomach.

Descending spinal projections from the rostral gigantocellular reticular nuclei complex

Electrophysiological and physiological studies have suggested that the ventral medullary gigantocellular reticular nuclei (composed of the gigantocellular ventralis and pars alpha nuclei as well as the adjacent lateral paragigantocellular nucleus; abbreviated Gi‐LPGi complex) provide descending control of pelvic floor organs (Mackel [1979] J. Physiol. (Lond.) 294:105–122; Hubscher and Johnson [1996] J. Neurophysiol. 76:2474–2482; Hubscher and Johnson [1999] J. Neurophysiol. 82:1381–1389; Johnson and Hubscher [1998] Neuroreport 9:341–345). Specifically, this complex of paramedian reticular nuclei has been implicated in the inhibition of sexual reflexes. In the present study, an anterograde fluorescent tracer was used to investigate direct descending projections from the Gi‐LPGi complex to retrogradely labeled pudendal motoneurons (MN) in the male rat. Our results demonstrated that, although a high density of arborizations from Gi‐LPGi fibers appears to be in close apposition to pudendal MNs, this relationship also applies to other MNs throughout the entire spinal cord. The Gi‐LPGi also projects to spinal autonomic regions, i.e., both the intermediolateral cell column and the sacral parasympathetic nucleus, as well as to regions of the intermediate gray, which contain interneurons involved in the organization of pelvic floor reflexes. Lastly, throughout the length of the spinal cord, numerous neurons located primarily in laminae VII–X, were retrogradely labeled with Fluoro‐Ruby after injections into the Gi‐LPGi. The diffuse descending projections and arborizations of this pathway throughout the spinal cord suggest that this brainstem area is involved in the direct, descending control of a variety of spinal activities. These results are in contrast with our observations of the discrete projections of the caudal nucleus raphe obscurus, which target the autonomic and somatic MNs involved specifically in sexual and eliminative functions (Hermann et al. [1998] J. Comp. Neurol. 397:458–474). J. Comp. Neurol. 455:210–221, 2003.

Increased expression of mu opioid receptors in animals susceptible to diet-induced obesity.

Stimulation of mu opioid receptors preferentially increases the intake of a high fat diet. In this paper we investigated whether there was a difference in the expression of mu opioid receptors between animals susceptible (Osborne-Mendel) or resistant (S5B/Pl) to obesity induced by eating a high fat diet. Immunohistochemical studies demonstrated that Osborne-Mendel rats eating a chow diet had an increased number of mu opioid receptors in the arcuate nucleus when compared to S5B/Pl rats. These immunohistochemical findings were supported by Real Time-PCR which demonstrated that the mRNA level of mu opioid receptors was also increased in the hypothalamus of Osborne-Mendel rats compared to S5B/Pl rats. Low doses of the mu opioid receptor agonist DAMGO [d-Ala(2)-N-Me-Phe(4)-Glycol(5)]-enkephalin administered to Osborne-Mendel rats caused a significant increase in the preference for a diet high in fat. The same doses of DAMGO switched the diet preference of S5B/Pl rats to high fat but did not significantly increase food intake. The combination of these findings suggests that the increased levels of hypothalamic mu opioid receptors in Osborne-Mendel rats may contribute to their preference for a diet high in fat and increase their susceptibility to becoming obese.

A critical re-evaluation of the specificity of action of perivagal capsaicin

Perivagal application of capsaicin (1% solution) is considered to cause selective degeneration of vagal afferent (sensory) C fibres and has been used extensively to examine the site of action of many gastrointestinal (GI) neuropeptides. The actions of both capsaicin and GI neuropeptides may not be restricted to vagal afferent fibres, however, as other non‐sensory neurones displayed sensitivity to capsaicin and brainstem microinjections of these neuropeptides induce GI effects similar to those obtained upon systemic application. The present study used immunohistochemical, biophysical and functional approaches to test the hypothesis that perivagal capsaicin induces degeneration of vagal efferents controlling GI functions. Our data indicate that perivagal application of capsaicin induces degeneration of vagal efferent motoneurones and decreased vagal motor responses. Treatment with perivagal capsaicin cannot therefore be considered selective for vagal afferent C fibres and, consequently, care is needed when using perivagal capsaicin to assess the mechanism of action of GI neuropeptides.

Physiology and mechanics of rat levator ani muscle: Evidence for a sexual function

The levator ani (LA) of male rodents is a classic model tissue for the study of hormone-muscle interactions, although its functions remain unknown. Recordings during copulation from chronic electromyographic (EMG) electrodes in the LA and bulbospongiosus (BS) revealed that EMG activity in the LA and BS was tightly coordinated. The LA was not active during noncopulatory behaviors, including the 1-min interval surrounding defecation. Electrical stimulation of the LA motor nerves increased penile bulb pressure. Increases in penile bulb pressure following BS nerve stimulation were markedly attenuated after LA denervation and were reduced further by LA removal. Stimulation of the LA nerve yielded insignificant changes in rectal pressure. Perineal motion analysis demonstrated that the LA acts upon the penile bulb and the surrounding BS exclusively. Apparently the rodent LA muscle is an active component in a highly coordinated neuromuscular system augmenting penile erection and, contrary to its name, is most unlikely to participate in alimentary function.

Descending projections from the nucleus raphe obscurus to pudendal motoneurons in the male rat

Previous physiological and behavioral studies have shown that the nucleus raphe obscurus (nRO) modulates pelvic floor reflex function (Yamanouchi and Kakeyama [1992] Physiol. Behav. 51:575–579; Beattie et al. [1996] Soc. Neurosci. Abstr. 22:722.4; Holmes et al. [1997] Brain Res. 759:197–204). In the present study, small injections of fluorescent tracers were used to investigate direct descending projections from the rostral and caudal portions of the brainstem nRO to retrogradely labeled pudendal motoneurons (MN) in the male rat. The caudal nRO projects into the ventral and lateral funiculi of the spinal cord, with arborizations in the thoracic intermediolateral cell column; in laminae VII, IX, and X of the lumbosacral cord; and in the sacral parasympathetic nucleus (SPN). Many identified external anal sphincter and ischiocavernosus MNs appeared to be in direct apposition with fibers originating from the caudal nRO; and more than half of the bulbospongiosus MNs that were identified appeared to receive such descending input. In addition to the nRO spinal autonomic and pudendal motoneuronal targets, projections were observed to regions of the intermediate gray that contain interneurons organizing the pelvic floor reflexes and to MN pools that are involved in functionally related somatic activities. Finally, several neurons in the lumbar enlargement were labeled retrogradely with FluoroRuby after injections into the nRO and the immediately adjacent reticular formation. Thus, the nRO may be in a position to modulate the coordinated actions of autonomic preganglionic and functionally related skeletal MN activity involved in sexual and eliminative reflex functions. J. Comp. Neurol. 397:458–474, 1998.

Serotonergic fiber sprouting to external anal sphincter motoneurons after spinal cord contusion.

The present study analyzed the anatomical plasticity of serotonergic immunoreactive projections to external anal sphincter (EAS) motoneurons, and the behavioral plasticity of EAS reflexes, penile erection, and locomotion in rats with spinal contusion injury (SCI) or complete spinal cord transection (TX). Electromyographic activity of the EAS, penile erection latency, and BBB locomotor score exhibited parallel recovery over the 6-week recovery period after contusion SCI. This pattern of recovery was not observed in TX animals. While locomotor scores demonstrated a small increase after TX, erectile and anorectal function remained at abnormal levels established immediately after injury. Serotonergic immunofluorescent (5-HT-IF) staining at the lesion site identified a small number of fibers spared after SCI that may provide a substrate for functional recovery. Pixel density measurements of 5-HT-IF in the vicinity of retrogradely labeled EAS and unlabeled pudendal motoneurons necessary for penile erection provide indirect evidence of serotonergic sprouting that parallels the observed functional recovery in animals with SCI. No 5-HT-IF was detected caudal to the injury site in TX animals. These studies indicate: (1) lumbosacral eliminative and reproductive reflexes provide a valid means of studying the mechanisms of post-SCI plasticity; (2) the similar recovery curves suggest similar return of descending control, perhaps through sprouting of descending serotonergic fibers; (3) the observed deficits after TX likely represent the permanent removal of descending inhibition and reflect reorganization of segmental circuitry.

Vagal afferent fibres determine the oxytocin-induced modulation of gastric tone

•  Oxytocin (OXT) inputs to the brainstem modulate cardiorespiratory, feeding and gastric functions. •  Vagal afferent (sensory) inputs are known to modulate brainstem synapses involved in visceral reflexes; however, the neurocircuits through which OXT exerts its actions are still unknown. •  In this study we elucidate these mechanisms of actions and report that vagal sensory fibres control these neurocircuits in a conditionally controlled manner such that brainstem synapses can prepare the neurocircuits to allow appropriate modulation of digestive processes. •  The results presented here improve our understanding of the central regulation of gastrointestinal functions and have the potential of being extended to the understanding of cardiorespiratory and feeding functions controlled by adjacent brainstem centres.

Effects of brain stem cholecystokinin-8s on gastric tone and esophageal-gastric reflex

The actions of cholecystokinin (CCK) on gastrointestinal functions occur mainly via paracrine effects on peripheral sensory vagal fibers, which engage vago-vagal brain stem circuits to convey effector responses back to the gastrointestinal tract. Recent evidence suggests, however, that CCK also affects brain stem structures directly. Many electrophysiological studies, including our own, have shown that brain stem vagal circuits are excited by sulfated CCK (CCK-8s) directly, and we have further demonstrated that CCK-8s induces a remarkable degree of plasticity in GABAergic brain stem synapses. In the present study, we used fasted, anesthetized Sprague-Dawley rats to investigate the effects of brain stem administration of CCK-8s on gastric tone before and after activation of the esophageal-gastric reflex. CCK-8s microinjected in the dorsal vagal complex (DVC) or applied on the floor of the fourth ventricle induced an immediate and transient decrease in gastric tone. Upon recovery of gastric tone to baseline values, the gastric relaxation induced by esophageal distension was attenuated or even reversed. The effects of CCK-8s were antagonized by vagotomy or fourth ventricular, but not intravenous, administration of the CCK-A antagonist lorglumide, suggesting a central, not peripheral, site of action. The gastric relaxation induced by DVC microinjection of CCK-8s was unaffected by pretreatment with systemic bethanecol but was completely blocked by NG-nitro-L-arginine methyl ester, suggesting a nitrergic mechanism of action. These data suggest that 1) brain stem application of CCK-8s induces a vagally mediated gastric relaxation; 2) the CCK-8s-induced gastric relaxation is mediated via activation of nonadrenergic, noncholinergic pathways; and 3) CCK-8s reverses the esophageal-gastric reflex transiently.

Dissociation of the effects of nucleus raphe obscurus or rostral ventrolateral medulla lesions on eliminatory and sexual reflexes

Rat preparations were used to investigate long-term changes in external anal sphincter (EAS) contractions and reflexive penile erection following electrolytic lesions of the nucleus raphe obscurus (nRO) or the rostral ventrolateral medulla. EAS contractions were measured electromyographically (EAS EMG) following distention of the EAS with a 5-mm probe. Penile erections were measured using a standard ex copula reflex testing paradigm. At 48 h postlesion, 100% of nRO-lesioned animals displayed reflexive erections and the magnitude of EAS EMG was significantly greater in lesioned animals than in sham controls. These results suggested EAS hyperreflexia following destruction of the nRO. By 14 days postlesion, EAS responsiveness in nRO-lesioned animals had returned to levels comparable to nonlesioned animals. No measures of penile erection were affected by nRO lesions. In animals with nucleus gigantocellularis (Gi) and lateral nucleus paragigantocellularis (Gi-lPGi) lesions, no significant changes to EAS reflexes were observed at any time point. At 48 h postoperative, Gi-lPGi lesions significantly reduced the latency to first erection and increased the number of erections elicited relative to controls. Similar facilitation of erection latency was observed at 14 days postlesion, while erection number and flip total were no longer significantly different from controls. These and previous studies suggest that the nRO regulates defecatory reflexes in the rat. These data further suggest that the comingled EAS and bulbospongiosus (BS) motoneurons are controlled by discrete and separate brainstem circuits and that increases in EAS and penile reflexes after spinal cord lesions are mediated by loss of different descending inputs.